Multiple sclerosis (MS) is a chronic disease of the central nervous system (CNS) characterized by local T cell and macrophage infiltrates, demyelination and loss of neurologic function [Steinman, 1996; Martin, et al., 1992; Mantzourani et al., 2005]. MS is an autoimmune disease triggered by CNS-specific CD4+ T lymphocytes. Candidate autoantigens include constituents of the myelin sheath, such as myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG).
An association between major histocompability complex (MHC) class II alleles and disease has been observed in MS patients, in particular HLA-DR1, HLA-DR2 and HLA-DR4. Although the pathology of MS remains unclear, there is evidence that T cells recognizing encephalitogenic epitopes of myelin, such as MBP, play a pathogenic role in the induction of MS. Studies have shown that T cell responses in patients are associated with the recognition of the 81-105 region of MBP (QDENPVVHFFKNIVTPRTPPPSQGK; SEQ ID NO. 4), and with highest affinity and binding to HLA-DR2 for the peptide epitope MBP83-99 (ENPVVHFFKNIVTPRTP; SEQ ID NO. 1). T cell recognition of this region of MBP has also been shown in healthy individuals, although at relatively low precursor frequencies. The binding of MBP83-99 to HLA-DR2 is via hydrophobic V87 and F90 residues, whilst, H88, F89, and K91 are TCR contact residues [Mantzourani et al., 2005].
The pathogenic role of autoimmune T cells recognizing encephalitogenic epitopes of MBP has also been noted in experimental autoimmune encephalomyelitis (EAE), one of the best-studied experimental animal models of MS. EAE represents an invaluable in vivo system for the evaluation of therapeutic approaches. EAE is induced in susceptible animals by immunodominant epitopes of the myelin sheath. Similar clinical and histopathological features to MS can be induced in susceptible mouse strains by immunization of myelin components. EAE is mediated by CD4+ T cells of the Th1 phenotype (IFN-γ). Like MS, EAE susceptibility is dependent on the MHC background of the mouse and different peptides are immunogenic and induce EAE in different strains. Myelin Oligodendrocyte Glycoprotein (MOG) residue 35-55 induces chronic (non-relapsing) EAE in C57BL/6 mice, guinea pig MBP residue 74-85 induces acute (relapsing-remitting) EAE in Lewis rats and proteolipid protein (PLP) residue 139-151 induces acute EAE in SJL/J mice [Zamvil et al., 1990].
The SJL/J mouse strain (H-2s haplotype) is commonly used for EAE since numerous histopathological, clinical and immunological features resemble that of human MS compared to other mouse or rat strains. In the SJL/J mouse strain, residues from the encephalitogenic epitope MBP81-100 have been shown to bind with high affinity. In fact, the minimum epitope required for binding is MBP83-99. Furthermore, in SJL/J mice, CD4 T cell responses to PLP residues 139-151 (PLP139-151: sequence HSLGKWLGHPDKF; SEQ ID NO. 3) is qualitatively different from responses to encephalitogenic of MBP83-99 in that the PLP peptide-specific clones are heterogeneous. As a first step toward understanding the cellular and molecular basis and the biologic relevance of this heterogeneity, studies have determined whether multiple overlapping epitopes within the PLP139-151 are responsible for the diversity. Initial studies demonstrated that the panel of T cell clones reacted with overlapping PLP peptides only when the peptides contained residue 144, thereby suggesting that this is an important site for the activation of all of the clones [Kuchroo, et al., 1992]. Furthermore, W144 is the dominant TCR contact residue, as substitution at position 144 with, alanine (A), or other hydrophobic residues, such as phenylalanine (F), abolishes the in vitro stimulatory activity of the peptide and such analogues do not induce EAE [Kuchroo, et al., 1992]. The single TCR antagonist peptide analogue (L144/R147), in which both of the major TCR contact residues are substituted, showed the maximum antagonist activity (L144/R147), in vitro and also gave the best inhibition of EAE [Kuchroo, et al., 1994].
In the C57BL/6 mouse strain, residues 35-55 from MOG protein have been found to be encephalitogenic [McFarlin, et al., 1973]. Disease is elicited by immunization with MOG35-55, resulting in a CD4+ T helper-1 (Th1)-cell response that attacks the myelinated areas of CNS [Zamvil and Steinman, 1990]. T cells supported by monocytes and activated microglial cells mediate inflammation and demyelination. B cells and antibodies are not critical for EAE induction in mice, although antibodies that bind to epitopes of MOG35-55 enhance demyelination in some models [Linington, et al., 1988]. In Lewis rats, epitope MBP74-85 has been identified as immunodominant for EAE induction. Moreover, peptide analogues based on the human MBP83-99 epitope have been found to suppress the EAE symptoms induced from the encephalitogenic MBP74-85 epitope. [Mendel et al., 1995; Tselios et al., 1999; Tselios et al., 2000a; Tselios et al., 2000b].
In the light of the above, the peptides MOG35-55, PLP139-151, MBP74-85 and MBP83-99 and their head to tail cyclic counterparts clearly represent a promising starting point for the design of altered peptide ligands and peptide analogues, which could be used to alter T cell responses in these animal models and thus lead to new therapeutic approaches against MS and other autoimmune diseases. Moreover, epitopes of MBP, PLP and MOG from multiple sclerosis patients, MBP82-100, [Ala86]MBP83-99, [Ala8]MBP83-99, [Tyr89]MBP83-99, MBP110-118, MOG97-108, PLP97-117, PLP185-206, PLP40-60, PLP190-209, PLP184-199, PLP80-88, PLP30-49, PLP180-199 and the like are recognized/presented by T cells/B cells from the peripheral blood of MS patients or are able to induce peptide-specific T cells responses in individuals [Greer, et al., 1997, Singh, et al., 2004, Greer, et al., 2004, Tsuchida, et al., 1994, Trotter, et al., 1998].
Current Peptide Therapies for MS
Current peptide therapies of MS include treatment with interferons (interferon beta-1α and interferon beta-1(3) and glatiramer acetate (copolymer-1) which is a synthetic protein comprised of the major amino acids Glu, Gln, Lys, Arg of MBP. These immunomodulators have been approved by the FDA for patients with relapsing-remitting MS. Interferons given by subcutaneous injection reduce the frequency, severity and duration of exacerbation, but their impact on preventing long term disability has not yet been established. In addition, side effects are common and consist of reactions at the injection site, fever, myalgia and flu-like syndrome. So far the reported benefits from the use of interferons and copolymers are marginal and the need for improved therapeutics is imperative.
Another approach under clinical investigation for autoimmune suppression is the oral administration of autoantigens. Orally administered antigens have been shown to suppress autoimmunity in animal models, including EAE, collagen and adjuvant-induced arthritis, uveitis and diabetes in the non-obese diabetic mouse. Low doses of oral antigen induce antigen-specific regulatory T-cells which act by releasing inhibitory cytokines such as TGF-β, IL-4, and IL-10 at the target organ. Thus, one can suppress inflammation at a target organ by orally administering an antigen derived from the site of inflammation, even if it is not the target of the autoimmune response. Initial human trials of orally administered antigen have shown positive findings in patients with MS and rheumatoid arthritis. A double-blind, placebo-controlled, phase III multi-centre trial of oral myelin in relapsing-remitting MS patients is in progress, as are phase II clinical trials investigating the oral administration of type II collagen in rheumatoid arthritis, S-antigen in uveitis and insulin in type I diabetes. This promising method allows for oral administration which is advantageous over previous treatments with interferons and copolymer-1. However, issues relating to the peptidic nature and cost of the administered substance renders the non-peptide mimetic approach, even in its infancy, an attractive goal to pursue. In this regard, our cyclic epitopes, which are more stable than their linear epitope counterparts, offer this advantageous property.
The present invention seeks to provide a new approach towards the therapeutic management of MS. More specifically, the invention focuses on the design and use of peptide analogues of disease-associated myelin epitopes to induce peripheral T-cell tolerance.